Dermal drug delivery system

ABSTRACT

A device for brushing the skin prior to applying a topical preparation of a drug enhances the permeability of the stratum corneum to the drug.

The present invention relates to the delivery of drugs by abrading theskin, and devices for use with such methods.

In order for therapeutic quantities of drug to penetrate the skin, thebarrier properties of the stratum corneum must be overcome. The stratumcorneum exhibits selective permeability and allows only relativelylipophilic compounds with a molecular weight below 400 Daltons to pass.

Methods of overcoming the barrier properties of the stratum corneum maybe divided into chemical, such as penetration enhancers andsupersaturated concentrations, and physical, such as iontophoresis, skinelectroporation, ultrasound and powder needleless injection, methods. Inthe case of macromolecules (>1000 Da) the use of physical methods hasbeen shown to be advantageous over chemical methods, and has led to asignificant increase in the number of topical/transdermal devices.

The mode of operation of these devices includes skin disruption andmicroneedles/microblades. Gerstel (1976) was one of the first todescribe a drug delivery device for percutaneous administration ofdrugs. The device consists of a drug reservoir and a plurality ofmicroneedles extending from the reservoir, for penetrating the stratumcorneum and epidermis to deliver the drug. This device is the forerunnerof many devices on the market today. A recent commercialisation of thismicroneedle technology is the Macroflux) microprojection array developedby Alza Corporation.

Most transdermal devices are currently based on patch technology, andproblems encountered include irritancy and poor adhesion. Most patchesonly deliver about 10% of the total dose, with the 90% of the drugremaining in the patch being discarded.

Known abrasion devices are relatively expensive and can only be operatedby trained personnel, thereby limiting their ability to be used bypatients. Since it is also a painful surgical procedure, anaesthesia isadministered beforehand.

WO 01/89622 describes the use of an applicator with an abrading surfaceon which the drug is coated.

We have now, surprisingly, found that the properties of the stratumcorneum can be substantially altered by a short period of brushing, suchthat therapeutically effective amounts of drug can pass thereacross.

Thus, in a first aspect, the present invention provides a method for theconditioning of skin to enhance transdermal delivery of drug, the methodcomprising continuous brushing for a period sufficient to reduce, orperturb, the barrier qualities of the stratum corneum.

In this respect, brushing affects the stratum corneum in such a manneras to facilitate, normally, and preferably temporarily, passage of adrug across it, by physically disrupting the barrier thereby to reducethe resistance to the drug's passage.

By “barrier qualities” is meant those properties of the stratum corneumthat inhibit the passage of drug. Reducing the barrier qualities is alsoreferred to herein as “enhancing permeability” and other related terms.

By “transdermal” is meant delivery of drug across the stratum corneum.In general, drugs delivered by such a route are intended for localconditions of the skin, and are not usually intended for systemicdelivery. Thus, the drug will cross the stratum corneum where, normally,it will then reach the required site of action.

Any drug may be employed in the present invention, the term “drug” hererelating to any pharmaceutically active substance capable of deliveryacross the stratum corneum in association with the method of the presentinvention. Suitable examples of drugs are given below.

The “continuous brushing” may be interrupted, if desired, but it isintended that the brushing continue for a generally consistent periodranging between about 10 seconds and 5 minutes, although longer orshorter periods may be employed, depending on the results to be achievedand the nature of the drug, as well as other factors, such as pressureapplied to the brush.

Thus, by “continuous” is meant that, between the start and finish of thebrushing process, the brushing should take place for greater than 50% ofthe time, preferably greater than 80%, and is, most preferably,substantially uninterrupted.

The nature of the brushing may be a simple backward and forwards action,but is preferably oscillatory, and is particularly preferably circularor rotatory. Not only is a circular brushing motion easy to arrange andemploy, but it has also been found that the circular motion results inthe greatest enhancement of the permeability of the stratum corneum.

Without being limited by theory, it is believed that the continuousbrushing motion, especially when circular, not only serves to stretchthe skin slightly, but also serves to warm the surface of the skin, bothof these actions serving to enhance penetration of drug.

What is particularly surprising is that this circular brushing is ableto increase, temporarily, the permeability of the stratum corneum tosuch an extent that it is even greater than such treatments as tapestripping, a process which is only generally applicable to isolated skinand which substantially destroys the integrity thereof. By contrast, thebrushing of the present invention only has a temporary effect onpermeability but, whilst the effect is achieved, the permeability israised substantially.

The drug may be applied in any suitable form. It is generally preferredto apply the drug in the form of a solution, gel, cream, emulsion,colloidal system, foam, mousse, suspension or ointment, and thepreparation may generally comprise further ingredients, such aspenetration enhancers and emollients.

Suitable drugs for use in accordance with the present invention include,but are not limited to, those in the following Table, eitherindividually or in combination: Type Of Drug Local antipruriticsCrotamiton Doxepin hydrochloride Mesulphen Polidocanol Localanaesthetics Amethocaine (Hydrochloride in solutions or creams, base ingels or ointments) Amylocaine (Hydrochloride) Benzocaine Bucricaine(hydrochloride) Butacaine Sulphate Butyl Aminobenzoate PicrateCincocaine (base, hydrochloride or benzoate) Dimethisoquin HydrochlorideDyclocaine Hydrochloride Ethyl Chloride Lidocaine Lignocaine MyrtecaineOxethazaine (Oxetacaine) Prilocaine Propanocaine HydrochlorideTetracaine Antihistamines Antazoline Chlorcyclizine HydrochlorideDimethindene Maleate Diphenhydramine Histapyrrodine IsothipendylHydrochloride Mepyramine Mepyramine Maleate Tolpropamine HydrochlorideTripelennamine Hydrochloride Triprolidine Hydrochloride CorticosteroidsAlclometasone dipropionate Beclomethasone dipropionate Betamethasonevalerate Clobetasol propionate Clobetasone butyrate DesoximetasoneDiflucortolone valerate Fludroxycortide/Flurandrenolone Fluocinoloneacetonide Hydrocortisone Hydrocortisone acetate Hydrocortisone butyrateTopical preparations for Calcipotriol psoriasis Coal tar Dithranol5-Fluouracil Ciclosporin Fumeric acid Lonapalene MethotrexateMethoxsalen Salicylic acid Tacalcito Tacrolimus Pimecrolimus TazaroteneTopical preparations for acne Azelaic acid Benzoyl peroxideDithiosalicylic acid Motretinide Resorcinol Topical antibacterials foracne Clindamycin Erythromycin ‘Dermatological drugs’ Becaplermin(Diabetic skin ulcers) Bentoquatum (prevents allergic contact dermatitiscaused by poison ivy) Gamolenic acid Glycolic acid (Photodamaged skin)Hydroquinone/Mequinol (Depigmenting agents) Ichthammol Keluamid(seborrhoeic dermatitis) Lithium succinate Monobenzone (vitiligo)Polyphloroglucinol Phosphate (Treatment of wounds and pruritic skindisorders) Sodium pidolate (humectant, applied as cream/lotion for dryskin disorders) Sulphur (mild antifungal/antiseptic) Sulphurated Lime(For acne, scabies, seborrhoeic dermatitus) Sulphurated Potash (Acne)Minoxidil (hair growth) Topical retinoids and related Adapalenepreparations for acne Isotretinoin Polyprenoic acid Tretinoin Othertopical preparations Nicotinamide for acne Topical antibacterialsAmphomycin Bacitracin/Bacitracin Zinc Bekanamycin SulphateChloramphenicol Chlorquinaldol Chlortetracycline Framycetin sulphateFusidic Acid Halquinol Mupirocin Mupirocin Neomycin sulphate Polymyxins(Polymyxin B Sulphate) Silver sulphadiazine (sulfadiazine)Sulphanilamide Sulphasomidine Sulphathiazole (sulfathiazole) SodiumTopical antifungals Benzoyl peroxide Amorolfine Benzoic acid BifonazoleBromochlorosalicylanilide Buclosamide Butenafine HydrochlorideChlormidazole Hydrochloride Chlorphenesin Ciclopirox OlamineClotrimazole Croconazole Hydrochloride Eberconazole Econazole nitrateFenticlor Fenticonazole Nitrate Flutrimazole Haloprogin KetoconazoleMepartricin Miconazole nitrate Naftifine Hydrochloride NatamycinNeticonazole Hydrochloride Nystatin Omoconazole Nitrate OxiconazoleNitrate Pyrrolnitrin Sertaconazole Nitrate Sodium Propionate SulbentineSulconazole nitrate Sulconazole Nitrate Terbinafine TioconazoleTolciclate Tolnaftate Triacetin Undecenoates/Undecanoic Acid Antiviralpreparations 1-Docosanol Aciclovir Brivudine Edoxudine IbacitabineIdoxuridine Idoxuridine in dimethyl sulfoxide Imiquimod PenciclovirVidarabine Parasiticidal preparations Benzyl benzoate Carbaryl MalathionPermethrin Phenothrin Preparations for minor cuts Cetrimide andabrasions Collodion Magnesium sulphate Proflavine Topical circulatorypreparations Heparinoid Antiperspirants Aluminium chlorideGlycopyrronium bromide

Other suitable drugs include the non-steroidal anti-inflammatories(NSAIDs), actinic keratosis treatments, and capsaicin.

The approach of the present invention may also be employed in thedelivery of protein and peptide substances, and related compounds, suchas peptidomimetics, and is of especial use in immunisation programmes,whether for primary or booster shots, and may be useful in BCG, forexample. The invention further extends to the delivery of nucleic acidsand their related compounds and derivatives. In the present context, aderivative or related compound is one that has been modified from theoriginal, or prepared independently from the original, and which is usedto emulate or to provide an affect associated with the original. Amimetic may simply comprise protecting groups, for example, or thebackbone may be modified to inhibit or block digestion of the molecule.

In particular, it has been found that the present invention isespecially suited for the delivery of hydrophilic drugs. The inventiondemonstrably increases the delivery rate of hydrophobic drugs, also, buthas an especially surprising effect with drugs having a Log P of ≦2, andis even more pronounced and beneficial with drugs having a Log P of ≦1,and especially with drugs having a Log P of 0 or below. For example,caffeine can be delivered at substantially increased rates using thepresent invention, and has a Log P of −0.07, as shown in Table 2, below.

Particularly preferred drugs for delivery using the present inventionare selected from; methotrexate, aciclovir, dactinomycin,oxytetracycline, 5-fluorouracil, ipatropium bomide, chlortetracycline,ceterizine, carboplatin, aminophylline, ofloxacin, pravastatin sodium,dichloromethotrexate, isoniaziad, theopylline, doxycyline,metronidazole, procaine, 4-aminosalicyclic acid, baclofen,triamcinolone, lidocaine/lignocaine, minoxidil, or combinations thereof.

The preparation of drug may be applied before, during, or after brushingthe skin, but the best effects are generally observed by brushing thearea of skin to be treated and then, substantially immediatelythereafter, applying the drug to the brushed area While it is notessential to apply the drug immediately after brushing, it will beappreciated that any substantial delay will permit the skin to recover,so that the enhanced permeability will be reduced, the longer the delay.

The nature of the brush is not critical to the present invention.However, it is preferred that the bristles making up the brush arepresented in a substantially planar fashion to the skin, although theremay be some advantage to having slightly longer bristles in the centreof the brush.

Where the bristles are presented in a substantially planar fashion tothe skin, then the area of skin over which the brush moves will begenerally equally treated, and there will be less discrepancy ofpermeation enhancement with increasing-pressure.

The bristles forming the brush should be sufficiently resilient as notto splay substantially under the pressures suitable to put the inventioninto effect. These pressures will generally range from about 20 g/cm² toabout 120 g/cm², and it will be appreciated that tougher, or moreresilient, bristles, are required with increasing pressure, in order toavoid substantial bristle deformation. With frequency of use, it islikely that a brush will start to deform, whereon it is generallydesirable to replace the brushing element. In any event, it isfrequently desirable to replace the brushing element between uses wherea device is intended for use on different patients. Where the patientapplies the treatment himself, then replacement may only be necessarywhen the brush remains deformed after use.

It is generally preferred that the bristles not be too hard, as this canlead to irritation and reddening of the skin which, whilst it may beacceptable for an occasional treatment, is undesirable where treatmentis required on a more frequent basis, such as daily, for example.

It has been found that the use of bristles having similar qualities tothose of hard brushes for use in domestic situations for application tothe person, such as nailbrushes, leads to very substantially increasedpermeation qualities of the stratum corneum, while not disrupting thestructure of the stratum corneum to any great extent, and that they canbe used without causing any substantial discomfort.

The stiffness of the bristle may also be referred to as the Robertsonnumber, and is commonly used in dentistry to define the hardness of abrush. A Robertson #6 is generally considered to be “soft”, and isusually at the low end of the range for what is suitable for use withthe present invention, given that such bristles deform under moderatepressure. To an extent, this can be accommodated by packing the bristlestightly, but is not generally as useful as using harder bristles.

In the accompanying Example, bristles of Robertson no. 8 provideexcellent results, without having to cause distress to the skin. It willbe appreciated that exceedingly hard bristles, such as wires, can beused, but these need to be employed with great care in order not todamage the skin, so that a maximum of a Robertson no. of 11 ispreferred.

The most preferred Robertson numbers are from about 6 to about 11, morepreferably from about 7 to about 10, particularly about 8 to about 9,especially about 8.

The duration of brushing is dependent on a number of factors, includingpressure on the brush, the hardness of the bristles and the nature ofthe drug to be delivered. However, it has generally been found that anappropriate length of time for brushing skin is between about 10 secondsand about two minutes, preferably between about 20 seconds and oneminute, and more preferably between about 30 seconds and about 50seconds, with an average of about 40 seconds generally providing a goodguide. With Robertson Grade 8 brushes, good efficacy is often observedat 15 seconds and above, and 25 seconds is often particularly effective.

The pressure on the brush should preferably be not so great as to causesubstantial irritation, but requires to be sufficient to have apermeabilising effect. As such, it has been found that a weight ofbetween 20 and 90 grams is generally effective, although, at 90 gramsand above, patient discomfort may be incurred.

In general, it has been established that a weight of about between 30grams and 80 grams is sufficient, with a weight of between 40 grams and60 grams being preferred. This weight is generally in terms of theaccompanying Example.

In terms of pressure, it is preferred to apply between about 20 gm/cm²and about 100 gm/cm², and more preferably between about 40 gm/cm² andabout 80 gm/cm².

The pressure applied to the brush may be any that is effective toenhance delivery of the selected drug or drugs across the stratumcorneum. In terms of N m⁻², particularly suitable pressures are about200 to about 1500 N m⁻². Pressures of greater than this may also beused, but generally require careful control, short duration, and higherbristle strength, so are not preferred. A more preferred range is about200 to about 1000 N m⁻², with pressures of about equal to, or greaterthan, 300 N m⁻² being especially preferred. A most preferred range isabout 300 to about 600 N m⁻².

The area of the end of the brush for contact with the skin may be assmall or as large as desired. For delivery of potent drugs, for example,it may be desired to apply the formulation in a diffuse fashion to alarger area, such as up to 500 cm², in which case it may be desirable tomove the brush over the skin to cover the whole of the target area,where the brush cross-section is smaller than the target area. It mayalso be desired to target a small area, or simply desired only to applyto a small area, to minimise any possible pain, so that an area of about1 mm² may be employed, for example. Smaller areas than this may provideproblems in finding bristles thin enough and strong enough to with standthe pressure while abrading the skin without causing damage. A smallrotating brash with short bristles will often suffice, under suchconditions. In general, an area of skin in the approximate region of 1cm² will often be the skilled physician's area of choice.

Thus, an area of the end of the brush of between about 1 mm² and about10 cm² is preferred, with a range of about 4 mm² to about 5 cm² beingmore preferred, and a range of about 5 mm² to about 2 cm² generallybeing sufficient for most applications.

The rate of oscillation, or rotation, of the brushes of the inventionmay be any that serves, in combination with other parameters, such asstiffness and cross-sectional area, to sufficiently perturb the skin toenhance passage of drug. In the accompanying Example, a rate of rotationof 80 revolutions per minute (rpm) was found to be effective. Rates downto 30 rpm may be employed, but will generally require stiffer bristlesand greater pressure to account for diminished interaction, while ratesof greater than 300 are increasingly associated with the risk of damageto the skin by treatment that is too harsh. Thus, rates of between 30and 300 rpm are generally preferred, with rates of 50 to 200 rpm beingmore preferred, and rates of 60 to 120 rpm generally being mostpreferred.

The present invention also extends to brushing devices for the skinadapted to enhance the permeability of the stratum corneum.

In one embodiment, the invention provides a mechanised brushing device,wherein the mechanism of the device is adapted to rotate a brush incontact with the skin of a patient, the device having abutment means forcontact with the skin, the brush being movably located in relation to”the abutment means to allow it to be introduced to the skin, travel ofthe brush being limited in relation to the abutment means such thatpressure on the brush to contact the skin is limited to a predefinedrange.

Such a device may be adapted to dispense drug during, or preferablyafter, brushing.

It will be appreciated that the term “mechanised” is used herein toindicate that the brush is not, directly, manually powered, but may bepowered by any suitable means, such as clockwork, solar-powered,battery-powered motor, electrically powered, or spring-loaded, forexample.

The brush will generally comprise a series of bristles which may beindividually mounted, or mounted in clusters, and which may be made ofany suitable material. It is preferred that the material be resilient orflexible, in order not excessively to abrade the skin. As a whole, thebristles should be sufficiently resilient to be load-bearing, in thateffective pressures may be applied through them without substantialdeformation at the recommended operating pressure for the brush. As thebristles will generally be resilient, some deformation under operatingpressure is to be expected. The brush will begin to lose efficiency whenparts of the bristle adjacent the terminus of the bristle, rather thanthe terminus itself, begin to impact the skin.

The material from which the bristles is made is not critical to theinvention, and many suitable bristle materials are well known in theart. Natural fibres, such as animal bristle, may be employed, but it isgenerally more convenient to employ synthetic polymers, such aspolyamides.

For example, a rotary battery powered brush may be spring mounted in anopen ended sleeve. Pressure on the housing causes the brush to movethrough the sleeve and to protrude beyond the opening, a tongue andgroove arrangement preventing the brush protruding too far. The openingin the sleeve abuts the skin such that, when the brush is at fullextension and the sleeve is in contact with the skin, then a weight ofsay 40 g is applied through the brush.

A simple timing mechanism may also be employed for example, to limit theduration to 40 seconds, for example.

One embodiment of this invention compromises a supporting body (rigidstructure, preferably made of plastic) from which an array/plurality ofbristles project vertically. The device is applied to the patient'sskin, in a position such that the tip of the bristles make contact withthe stratum corneum. A pre-wound spring is incorporated into thesupporting body, which when released by means of a control button(located on the supporting body) will allow the rotation/movement of thebristles in a clockwise or anticlockwise manner for a defined period oftime. The number of rotations can then be controlled by the energystored in the spring, and the manner in which the supporting body ismoulded can also control the pressure exerted on the skin. This allowsthe degree of attrition/perturbation/stretching of the stratum corneumto be performed in a reproducible manner. Another embodiment of thisinvention includes a system which will not only reduce the barriernature of the stratum corneum but also contain a formulation which wouldbe released at the same time.

In an alternative embodiment, a rotatably mounted brush is located in anextensible fashion in a body, with electrically powered motor meansbeing provided to rotate the brush. A battery may suitably provide theelectrical power. Actuation of the motor serves to engage the brush, andthis may be combined, such that extending the brush from the bodyactuates the motor. Preferably, a timer device cooperates with themotor, such that the motor switches off after a predetermined interval.The brush may then automatically retract, or may be manually orotherwise retracted.

The advantages offered by this system include the fact that it not onlyabrades the skin but also stretches it temporarily during treatment.This latter technique has been reported to enhance skin permeation(Cormier et al, 2001).

Using the rotating brush method achieves the purpose of reducing thebarrier nature of skin with little or no pain, compared to existingabrasion methods.

The ability of the device to enhance in vitro skin permeability wasinvestigated using the finite and infinite dose technique. Butylparaben, methyl paraben and caffeine were used as model penetrants dueto their similarity in molecular weight but differences inlipophilicity. Changes in the structural properties of the membrane werealso probed, using scanning and transmission electron microscopy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a shows the effect of rotating brush A on skin Permeation profileof CF at various pressures and durations;

FIG. 1 b shows the effect of rotating device brush A on skin Permeationprofile of BP at various pressures and durations;

FIG. 1 c shows the effect of rotating device brush A on skin Permeationprofile of MP at various pressures and durations;

FIG. 2 a shows the effect of rotating device brush A and B on skinpermeation of CF;

FIG. 2 b shows the effect of rotating device brush A and B on skinpermeation of MP;

FIG. 2 c shows the effect of rotating device brush A and B on skinpermeation of BP;

FIG. 3 shows the results of a pseudo-finite dose study comparing theeffect of a device of the invention using brush B with other establishedmodes of skin permeation enhancement;

FIG. 4 shows the effect of treatment time on the skin permeation profileof acyclovir using brush B;

FIG. 5 compares the effect of device treatment duration andiontophoretic treatment on skin permeation of radiolabelled ³H-labelledacyclovir after 60 min.; and

FIG. 6 compares the effect of a device of the invention, using brush Bwith other enhancement strategies on the skin permeation of Ang II.

The present invention will now be illustrated by the followingnon-limiting, Example.

EXAMPLE

A systematic investigation involving permeants of differinglipophilicity and molecular weight was performed to evaluate devices ofthe invention in enhancing skin permeation of permeants with differingphysicochemical properties.

Materials

The materials used in this Example were as listed in Table 1, below.TABLE 1 Materials used Materials and Equipment Supplier Methyl paraben(MP) Sigma Chemical Co., Dorset, UK Butyl paraben (BP) Caffeineanhydrous (CF) Triethylamine Angiotensin (Ang) II ³H-Acyclovir (ACV)Orthophosphoric acid BDH Laboratory Supplies, Potassium dihydrogenLoughborough, UK orthophosphate Methanol (HPLC grade) Rathburn ChemicalsLtd., Wakeburn, Acetonitrile (HPLC grade) Scotland Ethanol absolute(HPLC grade) Phosphate Buffered Saline Oxoid Ltd, Basingstoke, England(PBS) Deionised water (Elgstat Elga Ltd., High Wycombe, UK option 3A)Zovirax (5% Acyclovir cream) AAH supplies Enzyme immunoassay kitPeninsula Laboratories Inc., California, USA Liquid scintillationcocktail fluid Beckman Instruments Inc. California, USA Brush A: Softbristles Superdrug stores Plc, London, UK (Robertson Grade 6, brush headof electric toothbrush) Brush B: Hard bristles L. Cornelissen & Son Ltd,London, UK (Robertson grade 8, hog hair bristles) Motor (Type KQPS 22)Citenco Ltd, Borehamwood, Herts., UK

-   Brush Details-   Brush A-   Internally manufactured-   Oscillatory brush, Robertson (6) pure bristles-   Surface area of brush: 1.01 cm²-   Brush B-   Source: L. Comelissen & Son Ltd (London, UK)-   Brand name: Robertson (8) pure bristles-   Surface area of brush: 1.61 cm²

A summary of the properties of the model penetrants is given in Table 2,below, and a comparative description appears below. TABLE 2Physico-chemical properties of the model penetrants Penetrant MolecularWeight (Da) Log P (o/w) Butyl paraben (BP) 194.23 3.57 Methyl paraben(MP) 152.15 1.96 Caffeine (CF) 194.19 −0.07 Acyclovir (ACV) 225.21 −1.56Angiotensin (Ang) II 1046 —BP, MP and CF have similar molecular weights but differ inlipophilicity, thereby allowing the effect of the permeation enhancementmethod of the invention lipophilicity to be studied.ACV is an antiviral used in treating virus infections of the skin andmucosa. It is poorly absorbed across the skin due to its hydrophilicnature, hence reducing its therapeutic efficacy. ACV is marketed as acream and licensed to be used topically for the treatment of cold sores,however, due to its retarded diffusion across the skin, it isrecommended to be applied five times daily. Its dermato-pharmacologicalprofile therefore# makes it a useful model drug for testing the efficiency of thepermeation method or device of the invention. The use of ACV as a modelmarker in investigating the potential of enhancement strategies such asiontophoresis (Volpato at al., 1995, 1998; Stangi at al., 2004) andethosomes or ethanol based liposomes (Touitou at al., 2000) indecreasing the skin barrier to improve ACV diffusion has been reported.Ang II is a model hydrophilic, high molecular weight peptide which wouldnot ordinarily be absorbed across the skin by conventional formulationapproaches. Compared to the other model penetrants described above, itis the least likely to permeate the skin. Its use as a model marker toinvestigate the effect of iontophoresis# and jet injection methods on the in vitro permeability of largemolecular weight solutes across animal skin has also been reported(Clemessy at al., 1995; Sugibayashi at al., 2000).

The effectiveness of the device of the invention in increasing thepermeability of the above markers was also compared with some of theestablished and developmental methods of skin permeation enhancement,such as tape stripping, chemical enhancers, iontophoresis anddelipidisation (removal of skin lipids).

Analytical Methods

HPLC analysis of MP, BP and CF

Chromatographic measurements were carried out using a Perkin Elmer 200,LC Pump with Autosampler connected to a UW absorbance Detector 759A(Applied Biosystems, Foster city, Calif., U.S.A). The various HPLCmethods employed for each penetrant were shown to be fit for the purposeby ensuring reproducibility, repeatability, linearity, and intermediateprecision. Standard concentrations of the model penetrants (0.1-20μg/ml) were prepared in PBS and analysed. The chromatographic conditionsfor NT were as follows; Hypersil™ 5μ BDS, C18 (150×4.6 mm, 5 μm) column(Phenomenex® Ltd, Cheshire, United Kingdom) with the mobile phasecomprising 35% acetonitrile: 65% phosphate buffer (0.05 M KH₂PO₄containing 1% triethylamine, then adjusted to pH 3.5 withorthophosphoric acid). BP chromatographic conditions employed aLicrospher™, LispRP 18-5-1680, C18 (150×4.6 mm, 5 μm) column (Hichrom®Ltd, Berkshire, England), with a mobile phase of 50% acetonitrile: 50%phosphate buffer (0.02 M KH₂PO₄ adjusted to pH 3.0 with orthophosphoricacid). CF chromatographic conditions were as follows; Phenomenex®Prodigy™ 5μ ODS 2, C18 (150×4.6 mm, 5 μm), mobile phase comprising 90%acetonitrile: 10% phosphate buffer (0.02 M KH₂PO₄ adjusted to pH 3.0with orthophosphoric acid). Flow rate and injection volumes were set at1 ml/min and 10 μl respectively for all penetrants. Wavelength ofdetection was set at 254 nm, 256 nm and 270 nm for MP, BP and CFrespectively. Analytical determination of CF was modified wherenecessary to enhance its detection by increasing injection volume to 50μl and using a wavelength of 215 nm.

Analytical Studies for ACV

The radiochemical purity of the ³H-ACV to be used in this study wasdetermined by HPLC. Chromatographic measurements were carried out usinga Perkin Elmer, as described previously. ACV chromatographic conditionswere as follows; Phenomenex® Prodigy™ 5μ ODS 2, C18 (150×4.6 mm, 5 μm),mobile phase comprising 90% acetonitrile: 10% phosphate buffer (0.02 MKH₂PO₄ adjusted to pH 3.0 with orthophosphoric acid). Flow rate,injection volumes and wavelength of detection were set at 1 ml/min, 10μl and 250 nm respectively.

The purity of the ³H-ACV was determined by accurately spiking a 10 μlaliquot of the radiolabelled drug into 1.0 ml of PBS, which was theninjected. The purity of the radioactivity was determined by collectingsample fractions at 1 min time intervals from time 0 to 30 min.

Zovirax® spiked with ³H-ACV was used as the formulation for the entirestudy. Briefly, 300 μl of ³H ACV was placed into a 1.5 ml centrifugetube and evaporated off, over a stream of air. Approximately 1 g ofZovirax® was weighed into the centrifuge tube and carefully mixed with afine spatula to achieve a homogenous mix. To test for homogeneity, 3random samples (top, middle and bottom) were taken and determined forradioactivity via scintillation counting. This process was repeateduntil the % coefficient of variation (CV) was below 2.5%.

Analytical Development Studies for Angiotensin II

Analysis of the peptide was carried out using an enzyme immunoassay(EIA) as described in the EIA booklet. Briefly, a competitive enzymeimmunoassay which detects Ang II in biological matrices was employed.The principle of the assay is based upon the competition for antibodybinding sites between biotinylated and non-biotinylated peptides. Thebiotin group on the biotinylated peptide is then conjugated to SA-HRPwhich in turn reacts with another substrate (TUB) leading to theformation of colour. The absorbance recorded from each well is a directmeasure of the extent of binding for each peptide with the antibody.Absorbance was measured using a spectrophotometer. The method wasvalidated by calibrating the response at different concentrations of thepeptide (data not shown) and also the ability of the method to assessdifferent concentrations of the penetrant in the presence of anymatrices was investigated.

In vitro Skin Permeation Studies

Preparation of Human Epidermal Sheets

Human skin was obtained from cosmetic surgery with informed consent. Theepidermis was removed by the standard heat separation method (Khigmanand Christophers, 1963). Following removal of subcutaneous fat,individual portions of skin were immersed in water at 60° C. for 45seconds. The skin was then pinned, dermis side down, on a cork board andthe epidermis (comprising stratum corneum and viable epidermis) gentlyremoved from the under lying dermis. The latter was discarded and theepidermal membrane floated onto the surface of water and taken up onto aWhatman no. 1 filter paper. The resultant epidermal sheet werethoroughly dried and stored flat in aluminum foil at −20° C. until use.

Tape-Stripping Procedure

Tape-stripping was employed in order to partially remove the upperlayers of the skin where the barrier properties are known to reside.Tape-stripped skin sections used were prepared by repeated strippingskin with D-squame® adhesive disc. The disc was gently placed on theskin after which a known weight was placed into the adhesive disc skincomposite for 20 s. The weight was then lifted and the adhesive discremoved. This was then repeated eight times. The permeability profileacross the tape-stripped skin was then investigated via Franz cellstudies.

Skin Delipidisation Procedure

The objective here was to remove the intercellular lipids of the SC inorder to investigate the potential pathway employed by the model markersin their bid to cross the skin barrier. Delipidised skin was prepared byimmersing skin sections in chloroform and methanol (2:1) for. 40 minRastogi and Singh, 2001a,b), after which the skin was removed, blotteddry with tissue and dried via vacuum drier at 760 mm Hg, 25° C., for 1hr, to remove any remaining organic solvent. The delipidised skin wasthen used for Franz cell studies.

Chemical Enhancement Procedure (50% Ethanol in PBS)

The ability of ethanol to reduce the barrier property of the SC has beenwell documented (Williams & Barry, 2004). This was conducted by forminga saturated solution of each penetrant containing 50% v/v ethanol(EtOH), which was allowed to stir overnight. The resulting solution wasthen introduced into the donor well the Franz cells.

Iontophoresis or Post-Iontophoresis (Pre-Treatment)

This is a well established physical method of enhancing skin permeation,in vitro, which involves the use of electric current to permeabiliseskin and/or promote the migration of drug ions across skin (Cullander,1992; Guy et al., 2001; Kalia et al., 2004). Anodal treatment wasconducted by placing the anode electrode in the donor compartment andthe opposite electrode (cathode) in the receptor compartment of theFranz cell. Cathodal iontophoresis was conducted vice versa. The currenttreatment protocols employed were either

-   (a) iontophoresis: dose was administered simultaneously with current    or-   (b) post-iontophoresis: dose applied immediately after skin exposure    to current.-   A current intensity of 0.40 mA was employed for a 10 min period    during both protocols.    Novel Device (Rotatinig Brush) Procedure

The potential device parameters affecting permeation were identified asfollows:

-   (a) speed of bristles/frequency of movement;-   (b) pressure exerted on epidermal membrane surface;-   (c) duration of treatment; and-   (d) nature of bristle; hard or soft.

The mode of treatment was controlled as follows:

The piece of epidermal sheet with demarcated regions of interest was 1 aid on to a microscope slide which was then placed onto a weighingbalance.

The balance was supported by a jack (lift) sitting directly under thedevice the cell was then tarred.

With the aid of the jack, the device was partially lowered (byincreasing the height of the jack using the control knob) ensuring thatthe bristles did not touch the epidermal surface.

The device was then switched on, and the slide moved until a positionwas found where the bristles are directly under the demarcated region ofthe epidermal sheet.

The device was then switched off. The slide was then attached firmly tothe balance by means of scotch tape (tape extends from non demarcatedregion of epidermal sheet to balance, avoid contact between tape anddemarcated region to ensure no movement of the slide during treatment).

Once the slide was in a stationary position, the device was switched on,ensuring that the speed dial was at the required position. The devicewas gently lowered (by raising the jack as described above) until thebristles touched the surface, the reading on the balance then gave anindication of the weight (pressure) exerted on the membrane surface.

The required pressure was then attained by using the control knob of thejack to lower or increase the height of the balance. The slide thenremained stationary during the duration of treatment. Thepressure/weight exerted on the membrane during treatment was then readdirectly from the balance.

After treatment the scotch tape was carefully removed. The demarcatedcircular region was then cut off from the remaining epidermal sheet bymeans of a cork borer, then placed in a Franz cell.

An integrity check was also conducted after skin treatment to ensurethat the bristles did not create holes in the epidermal layer. This wasperformed by inversion of the Franz cell to observe whether the receptorfluid liquid was leaking through membrane.

Franz Cell Studies

Optimisation Studies using 3 Model Penetrants; Infinite Dose Study

The study was conducted using human epidermal sheets (since the barrierproperties reside in the stratum corneum) to determine the amount of themodel marker penetrating the membrane over a 4 hr period. CalibratedFranz cells of known area (˜0.65 cm sq) and volume (˜2 ml) were used.The receptor chamber was filled with PBS (pH 7.4) and stirred throughoutthe duration of the experiment by a PTFE coated magnetic flea. Themembrane was clamped in between the donor cap and receptor chamber ofthe Franz cell (stratum corneum facing upwards), was then treated withthe two types of bristles at different pressures and contact time,whilst maintaining a constant device speed (Table 3, below). TABLE 3Device parameters used during optimisation studies Speed (rpm) 80 Brush(bristle type) Soft (A) and hard (B) Pressure applied on skin (Nm⁻²) 300-1200 Treatment duration (s) 15-45

250 μl of saturated solution of the model penetrant in PBS was thendirectly introduced into the donor chamber of the cell. All experimentswere conducted in a water bath at 37° C. In order to determine theenhancement effect of the rotating brush, control experiments involvingthe use of non-treated (intact), tape stripped, donor solutions ofmarker containing 50% ethanol and delipidised epidermal membranes werealso performed.

Pseudofinite Dose-Studies (Acyclovir and CF)

Finite dose permeation experiments were performed using similarconditions as described above. The Franz cell studies were carried outwithout the use of a donor well, in order to allow for the placement ofthe iontophoretic device over the epidermal membrane. In addition, theepidermal membrane was mounted onto the receptor well usingcyanoacrylate adhesive and ensuring that there was no contact betweenthe adhesive and the effective surface area available for drugpermeation. After a drying time of 15 min, the receptor well was thenfilled with the receptor fluid (PBS). The effectiveness of the seal wasconfirmed if leakage of receptor fluid from the region of contactbetween skin and adhesive was not observed.

The skin was treated with the novel device using optimised parametersobtained from the preceding section of this Example. A formulation witha target dose of approximately 9±1 mg/cm² (ACV) and 20±2 mg/cm² (CF) wasapplied to the epidermal membrane surface using a previously calibratedpositive displacement pipette. Selected control experiments were thenperformed as described above (anodal and cathodal iontophoresis wasperformed by simultaneous application of dose and current over a 10 minperiod for CF and ACV respectively). At certain time intervals 200 μl ofthe receiver fluid was carefully withdrawn from the receiver fluid(maximum duration of 4 h). Approximately 4 ml of scintillation cocktailwas then added and the ACV sample analysed by scintillation counting,whereas the other penetrant was assayed via the HPLC method alreadydescribed.

Angiotensin II-Infinite Dose

A similar procedure described in the preceding optimisation section wasconducted for Ang II, whereby the diffusion of the peptide across skinfollowing brush treatment was compared against all the treatmentmethods. A 250 μl solution of Ang II in PBS (1 mg/ml) was thenintroduced into the donor well. The receptor chamber was filled with EIAbuffer, previously treated via ultrasound to prevent the formation ofair bubbles. Receptor fluid was sampled after a 4 h and 24 h period andanalysed via the EIA method described.

Histological Studies

The effect of bristle perturbation on the epidermis was assessed byscanning and transmission electron microscopy, where the integrity ofbrush treated and untreated (control) samples were compared. Brushtreated and untreated (control) samples were fixed in 2%formaldehyde/2.5% glutaraldehyde in 0.1M phosphate buffer pH 7.4overnight. Scanning electron microscopy (SEM) was performed as follows:A 5×5 mm square of skin was pinned to a thin piece of cork in order tokeep it flat during processing. Samples were immersed 20 min in each of30, 50, 70, 95, 100, 100, 100% v/v acetone in water, then dried usingliquid carbon dioxide in a Polaron E3000 critical point drier. Thesamples were then removed from the cork and mounted on 12.5 mm aluminumpin stubs using double sided adhesive carbon pads. Samples were sputtercoated with approximately 20 nm of gold in a Polaron E5100 sputtercoater and examined and photographed using a Philip SEM501B scanningelectron microscope. For transmission electron microscopy (TEM), a 1×2mm strip was cut from the fixed skin, then dehydrated by sequentialimmersion in acetone at increasing concentrations and embedded in spurrresin and polymerised for 48 hr at 60° C. Ultra-thin sections were cuton a Reichart Jung OMU4 ultra microtome using a diamond knife and thentaken up on 200 mesh hexagonal copper grids. Sections were stained for15 min in 1% w/w uranyl acetate in 50% v/v ethanol in water followed by5 minutes in Reynold's lead citrate. These sections were examined andphotographed using a JEOL JEM100CX II transmission electron microscope.

Data Interpretation & Statistical Analysis

Since the SC is made up of dead cells no active transport processesexist. Transport of the penetrant is therefore solely by passivediffusion, which can be described by Fick's first law (equation 1). Thediffusion of a drug across the stratum corneum (J_(S)) is therefore therate-determining step in skin permeation. $\begin{matrix}{J_{s} = \frac{D \times K \times C_{v}}{L}} & (1)\end{matrix}$

Where J_(S) represents the flux of the permeant across the membrane; Dis the diffusion coefficient of the penetrant in the membrane; K, thestratum corneum-vehicle partition coefficient; C_(v), the concentrationof the penetrant in the vehicle; and L the diffusional path lengthacross the membrane. J_(S) was determined from the linear portions ofthe permeation profile obtained from the infinite dose study as inaccordance with Fick's law. Permeability coefficient (K_(P)) which is aproduct of (D, K and L) was calculated as J_(S)/C_(v). The lag time(T_(L)) was determined by the intercept of the linear portions of theskin permeation profile on the x-axis where applicable. Enhancementfactors (EF) were calculated as a ratio of flux of permeant throughtreated skin to that of untreated skin. All data reported represent amean of n≧3-6 and its standard deviation (s.d.) or error (s.e.) exceptotherwise stated. Statistical analysis was conducted using the analysisof variance method (ANOVA) and student's t-test, the level ofsignificance was taken at p≦0.05.

Results and Discussion

Optimisation Studies

The infinite dose method was used to investigate the relationshipbetween the nature of bristles, pressure (weight) exerted on membraneand treatment time on permeation of the 3 markers. This was thencompared to already established methods of skin penetration enhancement.The rotational speed of the brush was maintained at 80 rpm for eachexperiment. The limitation of using the soft bristles (brush A) includedthe fact that the length of the bristles at the periphery of the brushwere slightly longer than those in the middle. Therefore, the surface ofthe brush was not uniform. Hence at low pressures (300 Nm⁻²) only theperipheral bristles were in contact with the skin, so that pressures≧450Nm⁻² were used, in order to ensure maximum contact with skin on usingbrush A. As a result of the flat nature of the surface of brush B (hardbristles) such problems were not experienced, thereby more readilyallowing the assessment of pressures at 300 Nm⁻².

Permeation of MP, BP & CF (Using Soft Bristles)

The human skin used in experiments involving brush A was from the samedonor. FIGS. 1 a-c show the effect of using the device with brush A(soft bristles) at different pressures and treatment times for thedifferent permeants. A minimum threshold pressure of 450 Nm⁻² and amaximum treatment time of 45 s was employed.

In FIG. 1 a, which shows the effect of rotating brush A on skinpermeation profile of CF: (⋄) untreated skin; (▪) 450 N m⁻², 20 s; (X)450 N m⁻², 45 s; (Δ) 750 N m⁻², 45 s; (♦) 1200N m⁻², 45 s. Datarepresents, mean±s.d. (n=3-6). Device speed maintained at 80 rpm

In FIG. 1 b, which shows the effect of rotating device brush A on skinpermeation profile of BP: (⋄) untreated skin; (▪) 450 N m⁻², 20 s; (X)450 N m⁻², 45 s; (Δ) 750 N m⁻², 45 s; (♦) 1200 N m⁻², 45 s. Datarepresents, mean+±s.d. (n=3-6). Device speed maintained at 80 rpm

In FIG. 1 c, which shows the effect of rotating device brush A on skinpermeation profile of MP: (⋄) untreated skin; (X) 450 N m⁻², 45 s; (♦)1200 N m⁻², 45 s. Data represents, mean±s.d. (n=3-6). Device speedmaintained at 80 rpm.

Where an enhancement factor (EF) of 2 or more was observed (i.e.significantly different from control) the use of a shorter treatmenttime of 20 s was investigated (c.f. CF—Table 4a and BP—Table 4b, below).The CF fluxes observed under conditions of 450 Nm⁻², 20 s, were howevernot significantly different from that of untreated skin. An increase intreatment time from 20 s to 45 s at constant pressure was found togenerally increase the flux of all the markers. The use of brush A wasfound to enhance the permeation of all the markers by at least anaverage factor of 2 (Tables 4a-c). Enhancement factors recorded were inthe order of CF≧BP≧MP. Increasing the pressure exerted on the membranewas found to enhance permeation. However, no significant differences(p≧0.05) were observed in flux between 450 Nm⁻² and 1200 Nm⁻² at atreatment time of 45 s. This may be due to the fact that, at weightsgreater than 450 Nm⁻², the nature of the bristles of brush A becomes alimiting factor, such that further increase in applied pressure did notyield an increase in flux. TABLE 4a Effect of treatment on in vitro skinpermeation parameters of CF using brush A J_(S) K_(P) T_(L) Skintreatment (10⁻⁴ μg/cm/s) (10⁻⁸ cm/s) (min) EF Untreated 6.96 ± 1.53 3.39 ± 0.75 77.66 ± 12.96 —  450 Nm⁻²/ 10.3 ± 2.67^(p) 5.04 ± 1.30 68.33± 17.76 1.47 20 s  450 Nm⁻²/ 16.3 ± 0.86* 7.95 ± 0.42 41.21 ± 8.09  2.3445 s  750 Nm⁻²/ — — — — 45 s 1200 Nm⁻²/ 18.4 ± 8.93* 8.97 ± 4.06 16.74 ±6.50  2.64 45 s Delipidised 52.25 ± 25.17* 25.66 ± 12.28 4.19 ± 1.397.47Data represents mean ± s.d. (n ≧ 3) except where otherwise stated.^(p)represents n = 2*Flux significantly different from that of untreated skin (p ≦ 0.05).Device speed maintained at 80 rpm.

TABLE 4b Effect of treatment on in vitro skin permeation parameters ofBP using brush A J_(S) K_(P) T_(L) Skin treatment (10⁻³ μg/cm/s) (10⁻⁵cm/s) (min) EF Untreated 3.80 ± 0.46  1.90 ± 0.23 33.47 ± 8.79 — 450Nm⁻², 20 s 6.60 ± 0.49* 3.30 ± 0.25 37.10 ± 2.26 1.74 450 Nm⁻², 45 s7.50 ± 0.75* 3.75 ± 0.37 25.22 ± 2.17 1.97 750 Nm⁻², 45 s 8.26 ± 0.89*4.13 ± 0.45 26.34 ± 1.48 2.17 1200 Nm⁻², 45 s 8.62 ± 1.05* 4.31 ± 0.5226.39 ± 1.30 2.26 Delipidised 15.95 ± 1.55*  7.98 ± 0.78 18.58 ± 3  4.20Data represents mean ± s.d. (n ≧ 3) except where otherwise stated.*Flux significantly different from that of untreated skin (p ≦ 0.05).Device speed maintained at 80 rpm.

TABLE 4c Effect of treatment on in vitro skin permeation parameters ofMP using brush A. J_(S) K_(P) T_(L) Skin treatment (10⁻³ μg/cm/s) (10⁻⁶cm/s) (min) EF Untreated 7.46 ± 0.67  3.83 ± 0.35 16.61 ± 5.15 —  450Nm⁻², 9.78 ± 0.54* 5.02 ± 0.27 16.12 ± 4.45 1.31 45 s  750 Nm⁻², — — — —45 s 1200 Nm⁻², 11.16 ± 1.60*  5.74 ± 0.83 12.43 ± 5.46 1.50 45 sDelipidised 28.40 ± 0.29*  14.59 ± 1.48   3.87 ± 2.09 3.81Data represents mean ± s.d. (n ≧ 3) except where otherwise stated.*Flux significantly different from that of untreated skin (p ≦ 0.05).Device speed maintained at 80 rpm.

The hydrophilic nature of CF results in very long lag times, as observedin this experiment (˜70 min) with untreated skin. The effect of thedevice on the lag times was profound in the case of CF, with alteringeither the pressure or duration of treatment significantly reducing thelag times by ˜75% of their original value. The use of delipidised skinwas also found to be more effective than use of the device with brush Ain enhancing permeation of the 3 markers. The lower EF's recorded forthe parabens, compared to CF, signifies the relative ease at which suchlipophilic markers permeate the SC, either in the absence or presence ofskin lipids.

The electron micrographs obtained (not shown) demonstrated somedisruption (attrition) of the Stratum corneum and the associatedloosening of these layers. Such perturbation possibly createschannels/disruptions in the membrane, which may account for the increasein flux of the permeants. Disruption is restricted within the upperlayers of the skin, whilst the remainder of the epidermis is unaffected.Enhancement factors less of 2 recorded in this part of the studyprompted the use of harder bristles (brush B) in order to furtherenhance skin permeation.

Permeation of CF, MP and BP using Harder Bristles (Brush B)

Surface analysis of the epidermis by electron microscopy showed that theextent of barrier disruption or perturbation induced by the device, onusing brush B, was slightly greater than that of brush A (data notshown)

Release profiles shown in FIGS. 2 a-c, (Data in Tables 5a-c, below)depict the effect of bristle type on skin absorption of the 3 markers. Aminimum threshold pressure of 300 Nm⁻² and a maximum treatment time of45 s was employed. Significant differences (p≧0.05) in penetrant fluxacross human epidermal sheets on using brush B relative to brush A wasobserved for all permeants.

In FIG. 2 a, which shows the effect of rotating device brush A and B onskin permeation of CF; (▪) untreated skin (□) brush A at 300 N m⁻², 45s; (+) brush B at 300 N m⁻², 15 s; (▴) brush B at 300 N m⁻², 25 s. Datarepresents, mean±s.d. (n=3-6). Device speed maintained at 80 rpm. Skindonor used for CF different from that used for MP and BP in FIGS. 2 b-c.

In FIG. 2 b, which shows the effect of rotating device brush A and B onskin permeation of MP; (▪) untreated skin (□) brush A at 300 N m⁻², 45s; (▴) brush B at 300 N m⁻², 25 s; (Δ) brush B at 450 N m⁻², 15 s. Datarepresents, mean±s.d. (n=3-6). Device speed maintained at 80 rpm.

In FIG. 2 c, which shows the effect of rotating device brush A and B onskin permeation of BP; (▪) untreated skin (□) brush A at 450 N m⁻², 45s; (+) brush B at 300 N m⁻²/15 s; (▴) brush B at 300 N m⁻², 25 s; (Δ)brush B at 450 N m⁻², 15 s. Data represents, mean±s.d. (n=3-6). Devicespeed maintained at 80 rpm.

The higher EF's recorded for brush B are likely to be attributed to thedegree of perturbation imposed on the membrane relative to brush A(Tables 5a-c, below) with CF being most markedly affected, with anincrease in EF of between 37 and 64. TABLE 5a Effect of treatment on invitro permeation parameters of CF using brush B J_(S) K_(P) T_(L)Penetrant Skin treatment (10⁻⁴ μg/cm/s) (10⁻⁸ cm/s) (min) EF CF^(X)Untreated  1.87 ± 0.26 9.15 ± 1.27 19.69 ± 3.54 — 450 Nm⁻², 45 s^(a) 8.52 ± 0.29* 41.61 ± 14.19 16.74 ± 3.99  4.55 300 Nm⁻², 25 s 120.92 ±42.55*⁺ 589.93 ± 207.62 — 64.44 300 Nm⁻², 15 s  70.19 ± 34.87*⁺ 342.4 ±170.1 — 37.41 450 Nm⁻², 15 s — — — — Delipidised  20.43 ± 5.50*⁺ 99.68 ±26.84 — 10.93 Tapestripped  35.5 ± 29.5*⁺ 173.4 ± 144.0 17.16 ± 9.1519.01 EtOH/PBS  28.98 ± 6.39*⁺ 141.4 ± 31.21 26.71 ± 9.26 15.46Data represents mean ± s.d. (n ≧ 3).*Flux significantly different from untreated skin (p ≦ 0.05).^(a)Represents brush A.⁺Flux significantly different from skin treated with brush A (p ≦ 0.05).^(X)Skin donor used for CF different from that used for MP and BP inTables 4b-c.Device speed maintained at 80 rpm.

TABLE 5b Effect of treatment on in vitro permeation parameters of MPusing brush B J_(S) K_(P) T_(L) Penetrant Skin treatment (10⁻³ μg/cm/s)(10⁻⁶ cm/s) (min) EF MP Untreated 10.90 ± 1.28  5.14 ± 6.06 15.02 ±3.74  — 450 Nm⁻², 45 s^(a) 13.53 ± 0.95*  6.38 ± 0.45 6.18 ± 1.34 1.24300 Nm⁻², 25 s 50.16 ± 9.25*⁺ 23.66 ± 4.36 3.29 ± 2.09 4.60 300 Nm⁻², 15s — — — — 450 Nm⁻², 15 s 62.34 ± 12.75*⁺ 29.41 ± 6.01 6.73 ± 3.63 5.72Delipidised 41.98 ± 2.05*⁺ 19.81 ± 0.96 — 3.85 Tapestripped 53.86 ±12.27*⁺ 25.41 ± 5.79 2.71 ± 1.61 4.94 EtOH/PBS 44.45 ± 7.19*⁺ 20.96 ±3.39 7.76 ± 5.56 4.08Data in Table 5b represents mean ± s.d. (n ≧ 3).*Flux significantly different from untreated skin (p ≦ 0.05).^(a)Represents brush A.⁺Flux significantly different from skin treated with brush A (p ≦ 0.05).

TABLE 5c Effect of treatment on in vitro permeation parameters of BPusing brush B J_(S) K_(P) T_(L) Penetrant Skin treatment (10⁻⁴ μg/cm/s)(10⁻⁵ cm/s) (min) EF BP Untreated  4.93 ± 0.22 2.47 ± 0.11 35.84 ± 1.98— 450 Nm⁻², 45 s^(a)  8.38 ± 1.09* 4.19 ± 0.54 24.51 ± 5.50 1.69 300Nm^(−2,) 25 s 10.47 ± 0.96* 5.23 ± 0.48 23.06 ± 2.19 2.12 300 Nm⁻², 15 s 6.16 ± 0.89* 3.08 ± 0.44 22.65 ± 3.22 1.25 450 Nm⁻², 15 s 12.69 ±2.07*⁺ 6.34 ± 1.03 14.55 ± 5.18 2.57 Delipidised 20.54 ± 2.85*⁺ 10.27 ±1.43   8.17 ± 5.41 4.16 Tapestripped 15.60 ± 3.69*⁺ 7.80 ± 1.84 34.78 ±4.44 3.16 EtoH/PBS 18.48 ± 1.61*⁺ 9.24 ± 0.80 26.43 ± 5.05 3.75Data in Table 5c represents mean ± s.d. (n ≧ 3).*Flux significantly different from untreated skin (p ≦ 0.05).^(a)Represents brush A.⁺Flux significantly different from skin treated with brush A (p ≦ 0.05).

The effect of brush B at 300 Nm⁻² was found to increase when treatmentduration was increased from 15 s to 25 s. Shorter treatment times andlower pressures were required in the use of brush B, when compared tobrush A, to produce significant changes to the barrier nature of the SCand, therefore, drug flux. With CF, significantly higher fluxes wereobserved with the use of brush B, compared to the other enhancementmethods, thus demonstrating the benefit of this device for suchmolecules. In the case of the parabens, the highest EF recorded on usingthe novel device was comparable to the established and developmentalmodes of skin permeation enhancement. Due to the remarkably high fluxesrecorded for CF with the use of brush B, the effect of increasingpressure exerted (>300 Nm⁻²) on CF flux was not investigated further.

CF Pseudo Finite Dose Study

This part of the study involved the use of the device with brush B andthe minimum and ideal conditions of pressure and a variation intreatment time to further evaluate, via the finite dose methodology, theeffect of the system using CF (FIG. 3). This was performed, in order toinvestigate the degree of enhancement provided-by the device underapplication conditions more like those that would occur in vivo. CF wasselected as a suitable candidate for this part of the study, due to itspoor intrinsic permeability across intact skin as well as for thepromising results recorded during the optimisation stages. For untreatedskin, and chemical enhancement procedures, the presence of CF was onlydetermined after 120 min (limit of detection of analytical method was0.05 ug/ml).

In FIG. 3, which shows the results of the pseudo-finite dose studycomparing the effect of the device using brush B (300 Nm⁻²/25 s) toother established modes of skin permeation enhancement; Mean±s.d.(n=3-6). *Amount of CF in receptor after 120 min. Device speedmaintained at 80 rpm.

Administered doses were found to dry into a thin film afterapproximately 1 hr. The amount of CF deposited in the receptorcompartment of the Franz cell, with the use of the chemical enhancementmethod, was found not to be significantly different (p≧0.05) fromuntreated skin. The amount of CF in receptor after 30 min using brush Bwas found to be significantly higher (p≦0.05) and at least double thatrecorded for the other methods of enhancement.

Permeation of ACV (Pseudo Finite Dose Study)

The poor efficacy of ACV topical preparations have been attributed itspoor skin permeability, resulting from its hydrophilicity, which hindersit from reaching the target site of the basal epidermis (Stagni et al.,2004). This, therefore, makes ACV an interesting model candidate for theevaluation of a novel transdermal device. The ability of the noveldevice to enhance the flux profile of ACV from a commercial cream isdemonstrated in FIGS. 4-5 and in Table 6.

In FIG. 4, which shows the effect of treatment time on the skinpermeation profile of acyclovir from a topical preparation (Zovirax®)using a device with brush B; (▪) untreated; (●) 10 s; (● with dashedline) 30 s; (▴) 60 s. Data represents mean+s.e. (n=4-9). Constant deviceparameters (speed; 80 rpm, pressure; 300 Nm⁻²).

In FIG. 5, which shows a comparison of the effect of device treatmentduration and iontophoretic treatment on skin permeation of radiolabelled³H-labelled acyclovir after 60 min. Mean 35 s.e. (n=4-9). Device speedmaintained at 80 rpm, pressure; 300 Nm⁻².

A significant increase in ACV flux across the skin was observed as thebrush treatment duration was increased, demonstrating the increasedbenefit of the device on delivering molecules like acyclovir across theskin. At maximum treatment time, the device of the invention resulted ina ca. 600% increase in enhancement factor, when compared toiontophoresis, also operated under optimum conditions (Volpato et al.,1995, 1998; Morrel et al., 2004). TABLE 6 Effect of using brush B atdifferent treatment times (10, 30 & 60 s) and iontophoretic treatment onskin absorption of radiolabelled acyclovir Amount in receptor after 60min (□g/cm²) EF Untreated 0.14 ± 0.08  — Brush treatment 10 s 5.06 ±1.88* 36 30 s 12.5 ± 4.02* 89 60 s 30.91 ± 5.45*  220 Iontophoresis^(c)4.95 ± 2.35* 35Data represents mean ± s.d. (n = 4-9) except otherwise stated.*Flux significantly different from that of untreated skin (p ≦ 0.05).Constant device parameters (speed; 80 rpm, pressure; 300 Nm⁻²).^(c)Cathodal iontophoresis (not post iontophoresis) performed for aperiod of 10 min, after which the current was switched off.Permeation of Angiotensin II (Infinite Dose Study)

The highly significant results in skin permeation enhancement observedfor the hydrophilic solutes ACV and CF prompted an investigation intothe ability of the device to enhance the permeation of a model peptide,Ang II. The hydrophilic nature and large molecular weight of the peptidemakes it an extremely unlikely candidate to be absorbed across the skin.

It can be seen from FIG. 6 and Tables 7a and b, below, that the use ofall the enhancement techniques, with the exception ofpost-iontophoresis, was found to significantly increase the permeationof the model peptide, compared to untreated (intact) skin at eachdefined time period (4 h and 24 h) although the effect was greatest withthe device of the invention.

In FIG. 6, which shows a comparison of the effect of a device fittedwith brush B with other enhancement strategies on the skin permeation ofAng II over a (□) 4 h and (▪) 24 h period. Data represents mean±s.d.(n=3-6).

The amount of Ang II permeating the skin was generally found to increasewith time, however, this increase was found not to be significant foruntreated, post-iontophoretic and tape-stripped skin. TABLE 7a Effect ofusing brush B and other treatment methods on the in vitro skinpermeation of Ang II after 4 h Amount in receptor after 4 h (ng/cm²) EFUntreated 0.27 ± 0.23 — Post-iontophoresis  0.46 ± 0.15⁺ 1.7Delipidisation 15.71 ± 2.85* 58 Tape-stripping 14.44 ± 4.73* 53 Rotatingbrush 19.45 ± 0.55* 72Data represents mean ± s.e. (n = 3-6) except otherwise stated.*Significantly different from that of untreated skin (p ≦ 0.05).⁺7.53 ± 5.87 ng/cm² was initially recorded (see FIG. 3), however thisamount was found not to be reproducible on sample re-analysis.

TABLE 7b Effect of using brush B and other treatment methods on the invitro skin permeation of Ang II after 24 h Amount in receptor after 24 h(ng/cm²) EF Untreated 1.69 ± 1.01  — Post-iontophoresis 10.02 ± 6.28  6Delipidisation 83.03 ± 24.28* 49 Tape-stripping 46.93 ± 15.62* 27Rotating brush 107.49 ± 19.66*  63Data represents mean ± s.e. (n = 3-6) except otherwise stated.*Significantly different from that of untreated skin (p ≦ 0.05).

Thus, the above Example clearly demonstrates the ability of theoscillating brush device of the invention to enhance in vitro skinpermeability and/or reduce lag times of permeants of differingphysicochemical properties across the skin, by the disruption of theupper skin layers. The surprising benefits of this approach whencompared to established and developmental methods of permeationenhancement, has also been clearly shown.

REFERENCES

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1. A mechanised brushing device, wherein the mechanism of the device isadapted to rotate a brush in contact with the skin of a patient, thedevice having abutment means for contact with the skin, the brush beingmovably located in relation to the abutment means to allow it to beintroduced to the skin, travel of the brush being limited in relation tothe abutment means such that pressure on the brush to contact the skinis limited to a predefined range.
 2. A device according to claim 1,further comprising pressure limiting means.
 3. A device according toclaim 1, adapted to provide a contact pressure of the brush of betweenabout 200 and about 1500 N m⁻² on the skin.
 4. A device according toclaim 3, wherein the pressure is between about 200 and about 1000 Nm⁻²,5. A device according to claim 4, wherein the pressure is about equalto, or greater than, 300 N m⁻².
 6. A device according to claim 4,wherein the pressure is between about 300 and about 600 N m⁻².
 7. Adevice according to claim 1, comprising a timing mechanism to limit theduration of the brushing.
 8. A device according to claim 1, adapted toprovide brushing for between about 10 seconds and 5 minutes.
 9. A deviceaccording to claim 8, adapted to provide brushing for between about 10seconds and about two minutes.
 10. A device according to claim 8,adapted to provide brushing for between about 20 seconds and one minute.11. A device according to claim 8, adapted to provide brushing forbetween about 30 seconds and about 50 seconds.
 12. A device according toclaim 1, wherein the brush is substantially Robertson grade 8, and isadapted to provide brushing for between about 15 seconds to about 40seconds.
 13. A device according to claim 1, adapted to provide anoscillatory brushing motion.
 14. A device according to claim 13, whereinthe oscillatory motion is essentially circular.
 15. A device accordingto claim 13, wherein the rate of said motion is between 30 and 300 rpm.16. A device according to claim 15, wherein the rate of said motion isbetween 50 and 200 rpm.
 17. A device according to claim 15, wherein therate of said motion is between 60 and 120 rpm.
 18. A device according toclaim 1, wherein that part of the brush for contact with the skinconsists essentially of bristles, and wherein said bristles have aRobertson number of from about 6 to about
 11. 19. A device according toclaim 18, wherein the Robertson number is from about 7 to about
 10. 20.A device according to claim 18, wherein the Robertson number is fromabout 8 to about
 9. 21. A device according to claim 18, wherein theRobertson number is about
 8. 22. A device according to claim 1, whereinthe cross-sectional area of that part of the brush for contact with theskin is between about 1 mm² and about 10 cm².
 23. A device according toclaim 22, wherein the cross-sectional area is about 4 mm² to about 5cm².
 24. A device according to claim 22, wherein the cross-sectionalarea is about 5 mm² to about 2 cm².
 25. A method for the conditioning ofskin to enhance transdermal delivery of drug, the method comprisingcontinuous brushing for a period sufficient to reduce the barrierqualities of the stratum corneum.
 26. A method according to claim 25,comprising, substantially immediately after brushing the area of skin tobe treated, applying said drug to the brushed area.
 27. A method for theconditioning of skin to enhance transdermal delivery of drug, the methodcomprising continuous brushing for a period sufficient to reduce thebarrier qualities of the stratum corneum, wherein said brushing isprovided by the mechanised brushing device of claim
 1. 28. A kitcomprising the mechanised brushing device of claim 1 and a drug whereinsaid drug is selected from the group consisting of: crotamiton, doxepinhydrochloride, mesulphen, polidocanol, amethocaine, amylocaine,benzocaine, bucricaine, butacaine sulphate, butyl aminobenzoate picrate,cincocaine, dimethisoquin hydrochloride, dyclocaine hydrochloride, ethylchloride, lidocaine, lignocaine, myrtecaine, oxethazaine, prilocaine,propanocaine hydrochloride, tetracaine, antihistamines, antazoline,chlorcyclizine hydrochloride, dimethindene maleate, diphenhydramine,histapyrrodine, isothipendyl hydrochloride, mepyramine, mepyraminemaleate, tolpropamine hydrochloride, tripelennamine hydrochloride,triprolidine hydrochloride, corticosteroids, alclometasone dipropionate,beclomethasone dipropionate, betamethasone valerate, clobetasolpropionate, clobetasone butyrate, desoximetasone, diflucortolonevalerate, fludroxycortide/flurandrenolone, fluocinolone acetonide,hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate,calcipotriol, coal tar, dithranol, 5-fluouracil, ciclosporin, fumericacid, lonapalene, methotrexate, methoxsalen, salicylic acid, tacalcitol,tacrolimus, pimecrolimus, tazarotene, azelaic acid, benzoyl peroxide,amorolfine, benzoic acid, bifonazole, bromochlorosalicylanilide,buclosamide, butenafine hydrochloride, chlormidazole hydrochloride,chlorphenesin, ciclopirox olamine, clotrimazole, croconazolehydrochloride, eberconazole, econazole nitrate, fenticlor, fenticonazolenitrate, flutrimazole, haloprogin, ketoconazole, mepartricin, miconazolenitrate, naftifine hydrochloride, natamycin, neticonazole hydrochloride,nystatin, omoconazole nitrate, oxiconazole nitrate, pyrrolnitrin,sertaconazole nitrate, sodium propionate, sulbentine, sulconazolenitrate, sulconazole nitrate, terbinafine, tioconazole, tolciclate,tolnaftate, triacetin, undecenoates/undecanoic acid, 1-docosanol,aciclovir, brivudine, edoxudine, ibacitabine, idoxuridine, idoxuridinein dimethyl sulfoxide, imiquimod, penciclovir, vidarabine, benzylbenzoate, carbaryl, malathion, permethrin, phenothrin, cetrimide,collodion, magnesium sulphate, proflavine, heparinoid, antiperspirants,aluminium chloride, glycopyrronium bromide, and mixtures thereof.
 29. Akit comprising the mechanised brushing device of claim 1 and a drugwherein said drug is selected from the group consisting of non-steroidalanti-inflammatories, actinic keratosis treatments, and capsaicin.
 30. Akit comprising the mechanised brushing device of claim 1 and a drugwherein said drug is a hydrophilic drug.
 31. A kit according to claim30, said drug having a Log P of ≦2.
 32. A kit according to claim 30,said drug having a Log P of ≦1.
 33. A kit according to claim 30, saiddrug having a Log P of 0 or below.
 34. A kit comprising the mechanisedbrushing device of claim 1 and a drug wherein said drug is selected fromthe group consisting of: methotrexate, aciclovir, dactinomycin,oxytetracycline, 5-fluorouracil, ipatropium bomide, chlortetracycline,ceterizine, carboplatin, aminophylline, ofloxacin, pravastatin sodium,dichloromethotrexate, isoniaziad, theopylline, doxycyline,metronidazole, procaine, 4-aminosalicyclic acid, baclofen,triamcinolone, lidocaine/lignocaine, minoxidil, and combinationsthereof.
 35. A kit comprising the mechanised brushing device of claim 1and a substance wherein said substance is a protein or peptide, nucleicacid, or a related compound.
 36. A kit according to claim 35, whereinsaid substance is capable of stimulating an immune response when appliedto the skin by a device as defined in claim 1.